The invention relates to a process for the in situ generation of amino acid chlorides utilizing bis-(trichloromethyl) carbonate, commonly known as triphosgene, and to methods of using this process for solid phase peptide synthesis and for derivatization of a solid support.
In the field of peptide synthesis certain couplings are known as difficult couplings, especially those involving coupling to bulky or sterically hindered amino acid residues, such as N alkylated, C-alkylated and Cxcex1branched amino acids. In order to obtain acceptable yields when these couplings are performed a variety of special coupling reagents have been developed. Among other known procedures, is the use of pre-formed amino acid chlorides to improve the outcome of the coupling reactions.
The general use of protected amino acid chlorides in solid phase peptide synthesis (SPPS) is limited mainly because of the fact that chlorides of fluorenylmethoxycarbonyl (Fmoc) amino acid having side chains protected with acid labile protecting groups, including but not limited to t-butyl (t-Bu), t-butoxycarbonyl (Boc) or trityl (Trt), have limited shelf stability. For example, chlorides of Fmoc-amino acids (AAs) with t-Bu-protected side chains could not generally be accommodated. In some cases (aspartic acid and glutamic acid) the chlorides could not be obtained and in other cases (tyrosine, serine, threonine) their shelf stability appeared insufficient for practical utilization. In addition, the preparation of chlorides derived from Fmoc- Lysine(Boc), Fmoc-Tryptophan (Boc), Fmoc-Cysteine(Trt), Fmoc-Glutamine(Trt) and Fmoc-Arginine 2,2,5,7,8-Pentamethyl chroman-6-sulphonyl (Pmc) is problematic because of side reactions and require special reaction conditions and purification (Carpino et al. Acc. Chem. Res. 29:268, 1996). This problem also hampers the general use of pre-formed Fmoc amino acid chlorides in automatic peptide synthesis. Despite these limitations, acid chlorides were used in SPPS especially for the assembly of hindered secondary amino acids (see Carpino et al. 1996 ibid and refs. within).
Coupling of protected amino acids to Nxcex1-alkylated amino acids was previously considered to be a difficult coupling both in solution and solid phase. This coupling was used in model peptides to demonstrate the efficiency of new, more effective, coupling methods. In these models, N-Methylated amino acids were used as nucleophiles, since coupling to N-Methylated amino acids having steric hindrance on the Cxcex1(e.g., N-methyl .valine and N-methyl Aminoisobutyric acid) was found to be much slower than to proline. Certain coupling agents and activation methods such as bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) (Coste et al. Tetrahetron Lett. 31 669, 1990), 1-hydroxy-7-azabenzotriazole (HOAt)/O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (Carpino et al. J Chem. Soc., Chem. Commun. 201, 1994), urethane- protected N-carboxyanhydrides (UNCA) (Spencer et al. Int. J. Pep. Prot. Res. 40:282, 1992) and acid halides (Carpino et al., 1996 ibid) were specially recommended to achieve coupling to N-alkyl amino acids.
The acid chloride method was found to be a superior way to couple protected amino acids to sterically hindered amino acid derivatives, such as the N-alkyl amino acids during SPPS of backbone cyclic peptides. To overcome the limitations of the pre-formed acid chloride method and to allow its general use in SPPS, it would be advantageous to have an efficient and generally applicable method allowing the in-situ generation of Fmoc-AAs chlorides.
The reagent bis-(trichloromethyl)carbonate (BTC) (Councler, C. Ber. Dtsch. Chem. Ges. 13:1697, 1880) also named hexachlorodimethyl carbonate or xe2x80x9ctriphosgenexe2x80x9d is a solid stable phosgene substitute equivalent to three moles of phosgene. Triphosgene has been used as an efficient carbonylating agent for liquid and solid phase synthesis of various aza-analogues of peptides containing aza-alanine, aza-aspartic acid and aza-asparagine residues (Andre et al. J. Pep. Sci. 3:429, 1997).
The use of triphosgene as a reagent for formation of isocyanates or other reactive species useful in peptide chemistry has also been disclosed (Eckert DE3440141, Nippon Kayaku JP10007623). The usefulness of triphosgene in preparation of various intermediates for pharmaceuticals has also been disclosed (Hoffmann et al. DD292452).
It is neither taught nor suggested in the art that the triphosgene reagent is suitable for the in-situ generation of protected amino acid chlorides, namely as a coupling agent in SPPS (for review see Cotarca et al. Synthesis 553, 1996)
Phosgene gas has long been a valuable asset to both lab and plant scale operations however the dangers of using it are also well documented, especially the respiratory hazards. Liquid trichloromethyl chloroformate, commonly known as xe2x80x9cdiphosgenexe2x80x9d (Fridgen, L. N. and Prol, J. J., J. Org. Chem. 54:3231, 1989) which has already been used as a phosgene substitute, has proven useful in all common phosgene reactions, but being a liquid its transport and storage still impose considerable hazard. Being a crystalline solid (mp 81-83xc2x0 C.), BTC is safer and easy to handle and therefore became the reagent of choice for all applications where phosgene chemistry is required (Cotarca et al. ibid). Synthetically, one mole of BTC yields three mole-equivalents of phosgene which reacts with hydroxyl, amine or carboxylic acid nucleophiles forming chloroformate, isocyanate or acyl chloride, respectively.
Considering all these features together with the fact that BTC is inexpensive and less susceptible to hydrolysis than phosgene, it is surprising that the use of BTC as a general coupling agent has not been considered.
Backbone cyclized peptide analogs
Backbone cyclization is a concept that allows the conversion of peptides into conformationally constrained peptidomimetics with desired pharmacological properties such as metabolic stability, selectivity and improved bioavailability (Gilon et al. Biopolymers 31:745, 1991; Byk et al. J. Med. Chem. 39:3174, 1996; Gilon et al. J. Med. Chem. 41:919, 1998). In backbone cyclization the Nxcex1 and/or Cxcex1 atoms in the peptide backbone are linked through various spacers. To synthesize N-backbone cyclic peptides a large number of orthogonally protected-finctionalized Nxcex1 alkyl amino acids (N- building units) were prepared (Bitan et al. J. Chem. Soc., Perkin trans. I 1501, 1997a; Muller et al. J. Org. Chem. 62:411, 1997). These units were incorporated into peptides by SPPS or solution methodologies and after orthogonal removal of the protecting groups from the xcfx89-functions on the Nxcex1-alkyl they are cyclized. A critical step in the synthesis of N-backbone cyclic peptides is the coupling of protected amino acids to the sterically hindered secondary amine of the Nxcex1 (xcfx89-functionalized alkyl) amino acid residue on the peptidyl-resin.
The synthesis of N-backbone cyclic peptides that incorporate Nxcex1 (xcfx89-functionalized alkyl) Glycine building units were reported previously. In these cases couplings of the protected amino acids to the secondary amine of the Gly building unit attached to peptidyl resin were achieved by multiple couplings with benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexaflourophosphate BOP or PyBroP as coupling agents (Bitan et al.. J. Pept. Res. 49:421, 1997b; Byk et al., 1996 ibid).
Generally, the coupling of protected AAs to building units other than Gly (non-Gly backbone cyclic building units) were found to be difficult and even impossible.
It has been shown that the coupling of many Fmoc AAs to sterically hindered secondary amines including a variety of non-Gly building units attached to peptidyl-resin could be achieved in moderate to high yields using the acid-chloride method but not acid fluorides (Carpino et al. 1996 ibid) or other coupling agents such as PyBrOP (Coste et al., ibid), HOAt/HATU Carpino 1994 ibid), 2-(2-Oxo-1(2H)-pyridyl)-1,1,3,3-bispenta-methyleneuronium tetrafluoroborate (TOPPipU) (Henkleinet al. In xe2x80x9cxe2x80x9cPeptides 1990xe2x80x9d Proc. of the 21th European Peptide Symposiumxe2x80x9d, E. Giralt and D. Andreu, eds, pp. 67. ESCOM Leiden, 1990), UNCA (Spencer et al., 1992) and Mukaiyama reagent (Mukaiyama, T. Angew. Chem., Int. Ed. Ingl. 18:7078, 1979).
The present invention relates to a process for the improvement of difficult couplings in SPPS. The invention also provides a method for improving the yield of the desired stereoisomer in solid phase peptide synthesis. In addition, the invention further provides methods for facilitating multiple parallel synthesis (MPS).
The present methods are useful for attaching protected amino acids to functionalized solid supports or to attach biomedically important ligands to a peptide or peptidyl resin during SPPS. Furthermore, these methods can be used to cyclize peptides attached to a solid support, or to form a urea bond in the sequence or in the bridge of cyclized peptides.
According to the present invention, a process is provided for the in situ generation of protected amino acid chlorides, by use of an agent such as phosgene, diphosgene or more preferably triphosgene. The protected amino acid chlorides thus generated are particularly useful in the coupling of an amino acid residue to a peptide chain. They can also be used for the coupling of a carbohydrate moiety to a peptide chain.
The in situ generation of acid chlorides using the methods of the present invention thus further provides a process whereby other biologically important acids, including but not limited to glucoronic acid, DTPA, and DOTA, may be connected to a peptide chain through the amine backbone or through an amino acid side chain functionality.
It has now been found that in accordance with the principles of the present invention bis-(trichloromethyl)carbonate, also known by the trivial chemical name triphosgene, can be used for the in situ generation of protected amino acid chlorides. This process overcomes many of the problems encountered in difficult coupling steps in SPPS, particularly where the coupling step involves sterically hindered amino acid residues or bulky amino acid analogs.
Methods are provided for the use of BTC as a convenient and efficient coupling agent for difficult couplings in SPPS. These methods provide greatly enhanced yields of the desired product in SPPS with retention of configuration and without undesired side reactions and also facilitate the performance of multiple parallel peptide synthesis. These methods also facilitate the synthesis of complex peptide analogs with multiple cyclizations, e.g., bi- and tri- cyclic peptides.
One method according to the present invention provides a process of coupling an amino acid residue to a peptide chain comprising:
(i) providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains;
(ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonate in an solvent inert to this reaction to obtain an amino acid chloride;
(iii) neutralizing the free acid by addition of an organic base;
(iv) adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a peptide having a blocked carboxyl terminus and a free amino terminus, and a peptidyl resin having at least one free amino terminus;
(v) providing reaction conditions enabling the coupling of the amino acid chloride to the peptide to yield a peptide elongated by one amino acid residue.
A second method according to the present invention provides a process of coupling an amino acid residue to a solid support comprising:
(i) providing an amino acid residue having a free carboxylic group and blocked amino group, optionally having additional blocked functional side chains;
(ii) reacting the blocked amino acid with bis-(trichloromethyl)carbonate in a solvent inert to the reaction to obtain an amino acid chloride;
(iii) neutralizing the free acid by addition of an organic base;
(iv) adding the resulting suspension containing the amino acid chloride to a compound selected from the group consisting of a resin having at least one free amino terminus and a solid support having a functional group capable of binding the chloride;
(v) providing reaction conditions enabling the coupling of the amino acid chloride to the solid support.
A currently most preferred embodiment according to the present invention is summarized below in Scheme 1: 
BTC=Bis-(trichloromethyl)carbonate (triphosgene)
Fmoc-AA=Fluorenylmethoxycabonyl-proteinogenic-xcex1-Amino Acid
R=Side chains of all proteinogenic xcex1-amino acids
Rxe2x80x2=CH3, (CH2)n=2-4-NH-Alloc, (CH2)n=2-3-COOAllyl
R3xe2x80x3N=tertiary or aromatic amine